ABO Blood GroupsBrian Poirier, MD

UCDavis Medical Center

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Topics

• Basic genetics of ABO blood groups

• Formation of H, A, and B antigens

• ABO antigens and antibodies

• ABO testing

• ABO discrepancies

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Objectives• Describe the inheritance of the ABO Blood

Groups and predict the ABO phenotypes and genotypes of offspring from various ABO matings

• Explain the roles of Secretor and H genes in the formation of H, A, and B antigens on the red cells

• Describe the reciprocal relationship between ABO antigens and antibodies for blood types O, A, B, and AB.

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Objectives (Continued)

• Describe the procedures on ABO forward and back typings; interpret the results; and resolve any discrepancies if present.

• Describe the quantitative and qualitative differences between the A1 and A2 antigens.

• Correctly identify all the ABO compatible blood components for each blood type

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Co-Dominance

1. A “Big” letter doesn’t mean dominant

2. A “small” letter doesn’t mean recessive

3. The exceptions to #1 and #2 are:– The Secretor genes: Se and se– The Lewis genes: Le and le– The H genes: H and h

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Inheritance of the ABO Blood Groups

• First described by Bernstein in 1924.

• A, B, O genes on chromosome #9

• The expression of antigens are based on the combination of three gene alleles: A, B, and O.

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Phenotypes vs Genotypes

Phenotypes Genotypes

Group O OO

Group A AA or AO

Group B BB or BO

Group AB AB

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ABO Phenotype Frequencies in U.S.

PopulationsPhenotype White Blk Mexican Asian

O 45 49 56 43

A1 33 12 22 27

A2 8 8 6 Rare

B 10 19 13 25

A1B 3 3 4 5

A2B 1 1 Rare Rare

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B Allele World Map

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Exercises

• Mother is type A and father is type O: What are the possible blood types for their offspring?

• Mother is type A and father is type B: What are possible blood types for their offspring?

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Basic Biochemistry

• Type I and Type II chains

• Se gene

• H gene

• Formation of the H antigen

• Formation of the A and B antigens

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Type I and Type II chains

Type I: primarily glycoproteins in secretions and plasma

• Saliva, clostrum, mothers’ milk, gastric fluid, bile, urine, serum, plasma, ovarian cyst

Type II: primarily glycolipids on RBCs• RBC, WBC, platelets, normoblast, sperm,

epidermal and epithelial cells

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Type 1: Secretor ABH Glycoprotein Substances

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Se Gene and Formation of the H Antigen

• Secretor = SeSe, Sese; Nonsecretor = sese• 80% of random population is either SeSe or

Sese• Secretor gene codes for fucosyl transferase• Enzyme (FUT2) adds fucose to type I

chains at terminal galactose; product is H antigen.

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H Gene and Formation of the H Antigen

• Phenotypes: HH, Hh, hh• Virtually 100% of random population is either

HH or Hh; hh genotype (lack of H =“Bombay phenotype”) is rare.

• H gene also codes for fucosyl transferase (FUT1)

• Enzyme (fucosyl transferase) adds fucose to terminal galactose of type II chains

• Final product is H antigen

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Formation of the H Antigen

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Type 2: H, A, and B Antigens

• H Ag: Gal–GlcNAc–Gal-X |

Fuc

• A Ag: GalNAc–Gal–GlcNAc–Gal-X |

Fuc

• B Ag: Gal–Gal–GlcNAc–Gal-X |

Fuc

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“A” Gene and Formation of the A Antigen

• H antigen is required for A antigen formation on RBCs or in secretions/plasma

• Formation of A antigen:

N-acetylgalactosamine is added to H antigen to make A antigen.

• A Ag

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Formation of the A Antigen

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“B” Gene and Formation of the B Antigen

• H antigen is required for B antigen formation on RBCs or in secretions/plasma

• Formation of B antigen: D-galactose is added to H antigen to make B antigen.

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Formation of the B Antigen

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The Residual H Antigen

• The more A or B antigen is made, the less H remains

• Relative amounts of H by blood group

O>A2>B>A2B>A1>A1B

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The Use of Lectins for Antigen Confirmation

• Dolichos biflorus = anti-A1

• Ulex europaeus = anti-H

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Questions?

• How is H antigen formed?– Relationship of Type I chain and Se gene – Relationship of Type II chain and H gene

• How are A and B antigens formed?• What blood type has the highest amount

of H antigen? What blood type has the least amount of H antigen? How would you determine that?

2804/19/23

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ABO Antigens and Antibodies

• ABO antigens based on combinations of three genes: A, B, and O

• Antibodies are clinically significant and “naturally occurring”– causing most fatal acute HTRs– some causing HDFN

• ABO antibodies neutralized with secretor saliva.

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Group O

• Generally the most common blood group• Genotype: OO• Antigen: H• Antibodies: anti-A, anti-B, and anti-A,B

– Antibodies are naturally occurring and very strong

– Anti-A,B (mostly IgG) may cross placenta to cause HDFN

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Group A

• Genotype: AA, AO• Antigen: A, H• Antibodies: anti-B (primarily IgM)• A subgroups

– A1 (80%) and A2 (20%) most important

– A1 has more A than A2 (quantitative difference); qualitative differences, too.

– ~25% of A2B’s form anti-A1

– 1-8% of A2’s form anti-A1

– Lectin of Dolichos biflorus agglutinates A132

Group B

• Genotype: BB, BO

• Antigen: B, H

• Antibodies: anti-A (primarily IgM)

• B subgroups: Not important

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Group AB

• Genotype: AB

• Antigen: A, B, very little H

• Antibodies: None

• B subgroups: Not important• A2B: 25% of A2B individuals

produce anti-A1

3404/19/23

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ABO Testing

• Cell typing (forward grouping) to determine antigen types on RBCs

• Serum/plasma typing (reverse grouping or backtyping) to determine type of antibody in serum:

• Note the opposite reactions– If the forward reactions are opposite of

reverse, an ABO discrepancy is not present.

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ABO Grouping Reagents

• Forward Grouping Reagent

• Reverse or Back Tying Cells

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Forward Grouping Reagent

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Forward Grouping

• Reagent: Monoclonal antibody– Highly specific– IgM– Expected 3+- to 4+ reaction– 1 drop– Anti-A=Blue; anti-B=Yellow (Acroflavin dye)

• A and B antigens on patient red cells are agglutinated by known sera (anti-A, anti-B)

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Reverse or Back Tying Reagent Cells

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Reverse or Back Typing

• Reagent Cells: Human Source– Expected 2+ to 4+ reaction– 4-5% cell suspension– 1 drop

• Anti-A or anti-B antibodies in patient serum (or plasma) agglutinate with A1 and B antigens on Reagent cells

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Forward Typing Procedures

• To determine what antigens are present on RBCs.

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Step 1. Label test tubes.

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Step 2: Make a 2-5% patient red cell suspension.

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Step 3: Add reagent antisera (1 drop).

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Step 3A: Add reagent Anti-A antisera (1 drop).

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Step 3B: Add Anti-B reagent antisera (1 drop).

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Step 4: Add one drop of 2-5% suspension of patient RBC to each

tube.

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Step 5: Mix and centrifuge (approximately 20 seconds).

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Group A: 4+ Agglutination with Anti-A

0 Agglutination with Anti-B

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Group B: 4+ Agglutination with Anti-B

0 Agglutination with Anti-A

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Group AB: 4+ Agglutination with Anti-A and Anti-B

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Group O:No Agglutination with Anti-A or

Anti-B

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Back Typing

• To determine what antibodies are present in patient’s plasma.

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Step 1: Label Test Tubes

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Step 2: Add two drops of patient serum to each tube

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Step 3: Add one drop of reagent cells to each test tube

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Step 3A: Add one drop of Reagent A1 cells

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Step 3B: Add one drop of Reagent B cells

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Step 4: Mix and centrifuge (approximately 20 seconds)

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Group A: 4+ Agglutination with B Cells

0 Agglutination with A1 Cells

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Group B: 4+ Agglutination with A1 Cells

0 Agglutination with B Cells

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Group O: 4+ Agglutination with A1 Cells

3+ Agglutination with B Cells

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Group AB:No Agglutination with A1 and B

Cells

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Exercises

• Interpretation of test results

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Exercises: Interpretation of ABO Testing Results

Forward Reverse Interpretation

anti-A Anti-B A1 cells B cells ABO Group

4+ 0 0 4+ ??

0 4+ 4+ 0 ??

4+ 4+ 0 0 ??

0 0 4+ 4 + ??

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Exercises: Interpretation of ABO Testing Results

Forward Reverse Interpretation

anti-A Anti-B A1 cells B cells ABO Group

4+ 0 0 4+ A

0 4+ 4+ 0 B

4+ 4+ 0 0 AB

0 0 4+ 4 + O

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What can Cause ABO Discrepancies?

• Disagreement between the interpretations of forward and reverse grouping

• Antigen problems

• Antibody problems

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Antigen Problems

• Lack of expected antigens– A subgroup– B subgroup– Bombay

• Presence of unexpected antigens– Acquired B phenotype – Polyagglutinable RBCs, recent marrow

transplant, nonspecific agglutination

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Antibody problems

• Lack of expected antibodies– Immunodeficiency, neonates, abnormally high

concentrations of Ab (prozone)

• Presence of unexpected antibodies– Anti-A1, cold auto-or alloantibodies, rouleaux

(false positive)

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A Subgroups

• A1

• A2

• A3

• Ax

• Aend

• Am

• etc

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A1 vs A2 Phenotypes

Blood Group Anti-A Anti-A1

lectin

A1 (80%) + +

A2 (20%) + 0

• A1 & A2 account for 99% of A group

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A1vs A2 Phenotypes

• Quantitative differences: More antigenic sites on A1 than A2.

• Qualitative differences between A1 and

A2 antigens:

– 1-8% of A2 individuals produce anti-A1

– 25% of A2B individuals produce anti-A1.

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B Subgroups

• Very rare and are less frequent than A subgroups.

• B subgroups demonstrate variations in the strength of the reaction using anti-B and anti-A,B

• Examples are: B3, Bx, Bm, Bel

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Acquired B phenotype

• Occurs in type A individuals with:

–Colon cancer, intestinal obstruction, gram negative sepsis

• Bacteria deacetylate group A sugar (GalNAc); remaining galactosamine crossreacts with reagent anti-B.

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Acquired B phenotype

Acquired B phenotype

• AB forward (with weak reactions with reagent anti-B)

• A reverse

• Reaction with anti-B is negative, if:

– Acidify serum

– Acetic anhydride treatment

– Auto incubation

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Acquired B typing result

Forward Reverse

Anti-A Anti-B Interp A1 cells B cells Interp

4+ 1-2+ AB 0 4+ A”B”

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Bombay (Oh) Phenotype

• Total Lack of H, A, and B antigens • Develop strong anti-H, anti-A, and anti-

B• “O” forward, “O” reverse; with positive

antibody screen• Require other Bombay donors for blood

transfusion• (“Para-Bombay” = H antigen in

secretions)80

Reactivity of Anti-H with ABO Blood Groups

O>A2>B>A2B>A1>A1B

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Blood Type: Antigens vs Antibodies

Blood Type Antigens Antibodies on rbcs in

PlasmaA A Anti-B

B B Anti-A

AB A,B None

O None Anti-A, Anti-B

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Exercise

Blood Compatible Compatible Compatible

Type RBCs FFPs Whole

Blood A ___ ___ ___

B ___ ___ ___

AB ___ ___ ___

O ___ ___ ___

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ABO Compatible Blood Components

Blood Compatible Compatible

Type RBCs FFPs A A, O A, AB

B B, O B, AB

AB AB, A, B, O AB

O O A, B, AB, O

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ABO Compatible Whole Blood

Blood type Compatible WB

A A

B B

AB AB

O O

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Consequences of ABO incompatibility

• Severe acute hemolytic transfusion reactions– One of the most frequent causes of blood

bank fatalities– Clerical errors

• Most frequent HDFN; usually mild.

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Sources of Technical Errors Resulting in ABO Discrepancies

• Inadequate identification of blood samples• Cell suspension too heavy or too light• Clerical errors• A mix-up in samples• Missed observation of hemolysis• Failure to add reagents• Failure to follow manufacturer’s instructions• Uncalibrated centrifuge• Contaminated reagents• Warming during centrifugation

8704/19/23

Resolving ABO Discrepancies

Problems with RBCs Resolution Techniques

Rouleaux wash RBCs 4X

MF agglutination check tx hx

Unusual phenotype (hh) Test with anti-H

Disease processes (Acq. B) check patient diagnosis

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Resolving ABO Discrepancies (Cont’d)

Problems with serum Resolution Techniques

Rouleaux Saline replacement

Presence of unexpected Ab Do panel to ID

Absence of expected Ab Increase incubation time

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Objectives

• Describe the inheritance of the ABO Blood Groups and predict the ABO phenotypes and genotypes of offspring from various ABO matings

• Explain the roles of Secretor and H genes in the formation of H, A, and B antigens on the red cells

• Describe the reciprocal relationship between ABO antigens and antibodies for blood types O, A, B, and AB.

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Objectives (Continued)

• Describe the procedures on ABO forward and back typings; interpret the results; and resolve any discrepancies if present.

• Describe the quantitative and qualitative differences between the A1 and A2 antigens.

• Correctly identify all the ABO compatible blood components for each blood type

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Reference Materials:1. Modern Blood Banking And Transfusion Practices, or 5th Edition

Denise M. Harmening. March 2005 . F.A. Davis. Philadelphia PA.2. Textbook of Blood Banking and Transfusion Medicine, Sally V.

Rudman. February 2005. W.B Saunders. Philadelphia PA.3. Transfusion Medicine Interactive: A Case Study Approach .

Marian Petrides MD, Nora Ratcliffe MD, and Roby Rogers MD. 2004. AABB Press Bethesda, Maryland.

4. Transfusion Reactions, 2nd Edition. Mark A. Popovsky (Editor). AABB Press 2001. Bethesda, Maryland.

5. American Association of Blood Banks Technical Manual (AABB) 14th Edition , 2003. American Association of Blood Banks, 8101 Glenbrook Road, Bethesda, Maryland.

6. Standards for Blood Banks and Transfusion Services 23rd Edition, 2004. Standards Committee, AABB. Bethesda, Maryland.

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